Agonist binding to β-adrenergic receptors on human airway epithelial cells inhibits migration and wound repair

Abstract

Human airway epithelial cells express β-adrenergic receptors (β-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the β2-AR-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the β-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by β-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that β-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-β1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with β-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in β1-integrin activation in the basal membrane.

β2-Adrenoceptor Agonists Promote Extracellular Signal-Regulated Kinase 1/2 Dephosphorylation in Human Airway Epithelial Cells by Canonical, cAMP-Driven Signaling Independently of β-Arrestin 2

“…Carvedilol, a partial β2-adrenoceptor agonist biased towards βArr2 (Liu et al, 2012;Wisler et al, 2007) was used for this purpose as it activates ERK1/2 in HEK293β2 cells (Luttrell et al, 2018;Wisler et al, 2007;this study). However, consistent with data gained in another epithelial cell line (Peitzman et al, 2015), carvedilol did not affect basal pERK1/2 levels in BEAS-2B cells. While this may indicate the absence of βArr2-dependent signaling, relatively low β2-adrenoceptor density (cf.…”

CFTR Involvement in Cell Migration and Epithelial Restitution

“…The reduced rate of migration following β 2 -AR activation was associated with a reduction in lamellipodia protrusion, similar in magnitude to the effect produced by CFTR channel inhibition with CFTR inh -172 or silencing by RNAi. Furthermore, β 2 -AR agonists, including carvedilol, decreased β 1 -integrin activation, and in CFTR-deficient Calu-3 cells, β 2 -AR activation had no effect on cell migration [114]. These findings suggested a model where exposure to β 2 -AR agonists stimulates PP2A phosphatase activity to produce dephosphorylation of multiple proteins involved in the control of cell motility (see Figure 3).…”

“…β 1 -integrin activation could be completely recovered by incubating CFTR deficient cells with exogenous GM1, but not with GM3 gangliosides, confirming that integrin activation was dependent on GM1 and CFTR expression. Reduced β 1 integrin phosphorylation was associated with lower levels of focal adhesion kinase (FAK) and Crk-associated substrate (CAS) phosphorylation which was [114] and [115]. also restored by incubation with exogenous GM1 ganglioside.…”

An in vitro model of chronic wounding and its implication for age-related macular degeneration

“…The integrity of the RPE monolayer along with the endothelial cells of Bruch's membrane are required to maintain the blood-retinal barrier [2]. To investigate the wound-healing capacity of differentiated human RPE, electric cell-substrate impedance sensing (ECIS) Zθ technology was utilized [39][40][41]. In this system, cells are grown on gold electrodes located in the bottom of an ECIS cultureware plate where the electrical impedance imposed by those cells is monitored and recorded by the application of a low voltage alternating current.…”